KRAS基因敲除A549细胞

The commonly mutated human KRAS oncogene encodes two distinct KRAS4A and KRAS4B proteins generated by differential splicing. We demonstrate here that coordinated regulation of both isoforms through control of splicing is essential for development of Kras mutant tumors. The minor KRAS4A isoform is enriched in cancer stem-like cells, where it responds to hypoxia, while the major KRAS4B is induced by ER stress. KRAS4A splicing is controlled by the DCAF15/RBM39 pathway, and deletion of KRAS4A or pharmacological inhibition of RBM39 using Indisulam leads to inhibition of cancer stem cells. Our data identify existing clinical drugs that target KRAS4A splicing, and suggest that levels of the minor KRAS4A isoform in human tumors can be a biomarker of sensitivity to some existing cancer therapeutics.

Kirsten rat sarcoma viral oncogene homolog (KRAS) oncogenic mutations are genetic drivers in various cancers, including non-small cell lung cancer (NSCLC). However, the regulatory mechanisms underlying the progression of NSCLC driven by oncogenic KRAS mutants are incompletely understood. Here, we show that ubiquitin specific peptidase 25 (USP25) impedes ring finger protein 31 (RNF31)-mediated linear ubiquitination of KRAS oncogenic mutants (KRAS) independently of its deubiquitinase activity, which facilitates the plasma membrane (PM) localization and the downstream oncogenic signaling of KRAS. Importantly, knockout (KO) of USP25 effectively suppresses tumor growth and RAS signaling in KRAS-driven autochthonous NSCLC mouse models and xenograft models, which is restored by additional deletion or inhibition of RNF31. Notably, knockin of USP25 in KRas-driven NSCLC models fails to inhibit cancer progression and reconstitution of USP25 into USP25 KO A549 cells restores tumor growth. These findings identify previously uncharacterized roles of USP25 and RNF31 in oncogenic KRAS-driven NSCLC progression and provide potential therapeutic targets for KRAS-related cancers.

BACKGROUND/OBJECTIVES:KRAS-mutated NSCLC has been targeted using monoclonal antibody (mAb) or tyrosine kinase inhibitor (TKI) therapies. However, in time, these mutations appear to develop resistance against the targeted antibodies and TKI treatments. One possible explanation is the activation of pro apoptotic pathways through the AXL-SRC-Akt axis. In this study, we identify AXL as the bypass resistant gene and investigate its role with KRAS and SRC activity. METHODS:In this study, we use Dasatinib and SGI-7079 to co-inhibit SRC and AXL respectively. In vitro studies were conducted using four cell lines, and AXL suppression was achieved using siRNA and in CRISPR-Cas9 mediated knockout models. Subsequently, we studied gene-protein expression analysis using Western blot, apoptotic markers using a cytochrome release assay and cytotoxicity using an MTT assay. A549 xenografts were studied for in vivo validation of our proposed hypothesis. RESULTS:The results suggest that dual inhibition of AXL and SRC significantly reversed this resistance, both in in vivo and in vitro studies. CONCLUSIONS:Co-inhibition of AXL and SRC synergistically reduced KRAS activity and induced apoptosis in NSCLC.